A cDNA gene (Auxyn10A), which encodes a mesophilic family 10 xylanase from Aspergillus usamii E001 (abbreviated to AuXyn10A), was amplified and inserted into the XhoI and NotI sites of pPIC9K(M) vector constructed from a parent pPIC9K. The recombinant expression vector, designated pPIC9K(M)-Auxyn10A, was transformed into Pichia pastoris GS115. All P. pastoris transformants were spread on a MD plate, and then inoculated on geneticin G418-containing YPD plates for screening multiple copies of integration of the Auxyn10A. One transformant expressing the highest recombinant AuXyn10A (reAuXyn10A) activity of 368.6 U/ml, numbered as P. pastoris GSX10A4-14, was selected by flask expression test. SDS-PAGE assay demonstrated that the reAuXyn10A was extracellularly expressed with an apparent M.W. of 39.8 kDa. The purified reAuXyn10A displayed the maximum activity at pH 5.5 and 50 °C. It was highly stable at a broad pH range of 4.5-8.5, and at a temperature of 45 °C. Its activity was not significantly affected by EDTA and several metal ions except Mn(2+), which caused a strong inhibition. The K(m) and V(max), towards birchwood xylan at pH 5.5 and 50 °C, were 2.25 mg/ml and 6,267 U/mg, respectively. TLC analysis verified that the AuXyn10A is an endo-β-1,4-D-xylanase, which yielded a major product of xylotriose and a small amount of xylose, xylotetraose, and xylopentose from birchwood xylan, but no xylobiose.

Abstract. hly is a cDNA gene derived from human leukocytes that encodes a mature human lysozyme (abbreviated to hLY). The aim of the present study was to determine the effect of cloned hly on recombinant hLY (r-hLY) activity under optimized conditions. hly was amplified by RT-PCR and ligated into the pPIC9K plasmid. The cloned cDNA (hly) was 393 bp in length, encoding a 130 amino acid hLY with a calculated molecular mass of 14,698 Da. The recombinant expression plasmid, designated as pPIC9K-hly, was linearized with SacⅠ and transformed into Pichia pastoris GS115 (his4, Mut + ) by electroporation. The integration of hly into the P. pastoris genome was confirmed by PCR analysis using 5'-AOX1 and 3'-AOX1 primers. Yeast extract peptone dextrose (YPD) plates containing different concentrations of geneticin (G418) were used for the screening of P. pastoris transformants (His + , Mut + ) with multiple hly copies. One transformant resistant to 4.0 mg/ ml of G418, designated as P. pastoris GShLY4-6, expressing the highest r-hLY activity was selected by the shake-flask test, and used for the optimization of expression conditions. When the P. pastoris GShLY4-6 was induced under optimized conditions, the expressed r-hLY activity was up to 533 U/ml, which was 1.52 times as high as that (351 U/ml) expressed using the standard protocol. SDS-PAGE assay demonstrated that the r-hLY with an apparent molecular mass of approximately 14.7 kDa was extracellularly expressed in P. pastoris. In conclusion, r-hLY increased following the cloning of hly and the optimized conditions as compared to standard protocol. IntroductionHuman lysozyme (EC 3.2.1.17) is a bacteriolytic enzyme that is widely distributed in a variety of tissues and body fluids (1). It hydrolyzes preferentially the β-1,4 glycosidic linkages between the N-acetylmuramic acid and N-acetylglucosamine residues that occur in the peptidoglycan cell wall structures of certain microorganisms, particularly those of Gram-positive bacteria, and therefore appears to have a role in host defense (2). The hLY comprises 130 amino acid residues and belongs to the group of lysozymes called c-type lysozymes (3). Due to its bacteriolytic activity, it is considered to be useful for medical and food uses. There is a reemergence of uses of lysozyme in various clinical trials. The addition of lysozyme to baby formulas was proposed (4), and lysozyme had also been shown to inhibit the growth of HIV-1 in vitro. Of note, the anti-HIV activity of lysozyme is independent of its muramidase activity (5). In addition, findings of previous studies have shown that it is effective in inactivating spores of Bacillus cereus in cheese by causing high hydrostatic pressure following the addition of lysozyme (6).Currently, hLY is mainly extracted from human milk and placenta, which is restricted by many factors, such as the lack of raw materials and high cost of purification. Therefore, the hen egg-white lysozyme is the most readily available form of commercial lysozyme, since it is relatively inexpensive compared...